Cyclic AMP stimulates the cyclic GMP egression pump in human erythrocytes: effects of probenecid, verapamil, progesterone, theophylline, IBMX, forskolin, and cyclic AMP on cyclic GMP uptake and association to inside-out vesicles.
The knowledge about the structure and function of the protein families responsible for cGMP synthesis and metabolic conversion has grown vastly the last years, whereas little is known about proteins that account for the cellular export of cGMP. In the present study, we have employed a model with inside-out vesicles prepared from human erythrocytes to characterize modulation and regulation of cellular cGMP extrusion. The active transport was saturable (Km of 2.4 +/- 0.2 microM, mean +/- SEM, n = 3) and coupled to ATP hydrolysis since no accumulation was detected in the presence of ATP-gamma-S and AMP-PNP. The observation that 100 microM of cAMP caused a minimal inhibition (14.4 +/- 0.3%) of active cGMP transport showed that the extrusion system for cGMP was not shared with cAMP, but a competitive interaction occurred for the ATP-independent association to the inside out vesicles. In contrast, the lowest, but physiological relevant cAMP concentrations (0.1-5 microM) stimulated the active cGMP transport with 30-35%, an observation that suggests cAMP as an allosteric regulator of the cGMP transporter. Several well-known modulators of other energy-requiring membrane transport systems caused a competitive and concentration-dependent inhibition, including verapamil (Ki = 13.0 +/- 2.4 microM), forskolin (Ki = 13.5 +/- 1.4 microM) and probenecid (Ki = 27.0 +/- 1.3 microM). Progesterone, which was the most potent inhibitor (Ki = 2.2 +/- 0.3 microM), interacted with the active cGMP transport in a noncompetitive manner. The highest concentration (100 microM) of IBMX and theophylline reduced the active cGMP uptake with 29.5 +/- 1.9% and 21.6 +/- 2.1%, respectively. None of these substances interfered with the association of cGMP to the vesicles in absence of ATP. The present results show that human erythrocytes possess a cell membrane cGMP transporter which is coupled to an ATPase. Its activity is regulated by cAMP in an apparent allosteric manner and inhibited by substances previously known to interact with other membrane transport systems.
Schultz
C
C
Department of Pharmacology, University of Tromsø, Norway.
Vaskinn
S
S
Kildalsen
H
H
Sager
G
G
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D001693:Biological Transport, Active / Q000187:drug effects; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000494:pharmacology*; D006152:Cyclic GMP / Q000378:metabolism*; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004910:Erythrocyte Membrane / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D016623:Ion Pumps / Q000187:drug effects / Q000378:metabolism*; D011339:Probenecid / Q000494:pharmacology; D011374:Progesterone / Q000494:pharmacology; D013806:Theophylline / Q000494:pharmacology; D014700:Verapamil / Q000494:pharmacology
D016623:Ion Pumps; D005576:Colforsin; D011374:Progesterone; D013806:Theophylline; D014700:Verapamil; D000242:Cyclic AMP; D006152:Cyclic GMP; D011339:Probenecid; D015056:1-Methyl-3-isobutylxanthine; D004121:Dimethyl Sulfoxide